FAAH, the fatty acid amide hydrolase, is responsible for the termination of the action of anandamide, an endocannabinoid. The overall goal of this proposal is to understand the mechanism by which FAAH regulates the levels of anandamide. The first Specific Aim is to characterize the mouse FAAH gene promoter elements responsible for FAAH expression in the brain and in other tissues. The activity of FAAH is regulated between cells, between organs, and by hormones and our research is designed to elucidate the mechanism by which this occurs. The second Specific Aim is to explain how FAAH, an intracellular enzyme, terminates anandamide's action by driving its cellular uptake. Anandamide is inactivated after being transported into cells and we postulate that FAAH contributes to anandamide uptake by creating and maintaining an inward concentration gradient. We will undertake FAAH inhibitor studies with analogs of methylarachidonyl fluorophosphonate (MAFP). We will employ various cell lines including primary cultured neuronal cells (striatal and cortical) from FAAH knockout and FAAH wild type mice. FAAH is a target for the design of therapeutic drugs since its inhibition (a chemical knockout) will raise the levels of extracellular anandamide at cannabinoid receptors. These studies have practical applications in the field of drug abuse where it would be clinically desirable to raise the levels of the endocannabinoids (e.g., during cannabinoid or opiate withdrawal). We will test the selectivity of a series MAFP analogs that inhibit FAAH towards five other enzymes. In the 3rd Specific Aim, the expression of FAAH and uptake of anandamide will be characterized in a model system (the mouse retina) where the cellular organization is very well characterized. We will determine if the transport of anandamide is greatest in retina cells that contain FAAH to validate the results of our cell culture experiments in Specific Aim 2. We will employ FAAH retina from knockout and FAAH wild type mice to undertake the first anandamide uptake studies in intact tissues where the individual cells can be identified. The long-term objective of the proposed research is to understand the role that FAAH plays in the inactivation of the endocannabinoids.